{"paper_id":"1e3acadd-b40c-4928-bfc7-8a12eb912d2e","body_text":"Endometriosis is a multifactorial disease which is characterized by presence and growth of endometrial glands and stroma outside the uterine. It affects approximately 5-10% of reproductive age women in USA ( 1 - 3 ). Eidences of immunological, environmental, endocrine and genetic factors involved in endometriosis pathogenesis are available, however, its etiology and pathogenesis remain unknown ( 4 ,  5 ). Contribution of genetic factors to endometriosis pathogenesis was confirmed by higher risk of endometriosis among monozygotic twins compared to dizygotic twins as well as higher risk of recurrence in first-degree relatives ( 5 ,  6 ).\ngenome-wide association studies showed that there is remarkable consistency in endometriosis ( 7 ). Several candidate genes in endometriosis including phase I and II detoxification genes, sex steroid pathways, cytokine signaling pathways, adhesion molecules and growth factor have been associated with endometriosis ( 8 ,  9 ). The glutathione S-transferases (GSTs) are the key phase II xenobiotic-detoxifying enzymes, which are upregulated in response to oxidative stress and overexpressed in many tumors ( 10 ,  11 ). The  GSTs  gene family encodes proteins that are critical for certain life processes, detoxication and toxification procedures. It is done by conjugation of reduced glutathione (GSH) with numerous substrates such as pharmaceuticals and environmental pollutants ( 11 ).\nTo date, the most significant evidence linking specific polymorphisms to endometriosis comes from studies investigating phase II detoxification enzymes ( 9 ).  GSTM1  and  GSTT1 , two member of GST gene family, have null allele variants, which their homozygosity causes a complete lack of enzyme activity ( 12 ,  13 ). A consistent association of a GSTT1 polymorphisms and endometriosis, with a 29% increased risk of endometriosis in  GSTT1  null deletion carriers has been reported ( 9 ). The  GSTP1  313 A/G polymorphism would result an amino acid substitution from isoleucine to valine at codon 105, which modifies the catalytic activity and heat stability of the enzyme ( 14 ).\nAccording to our knowledge, the results of  GSTP1  313 A/G polymorphism and  GSTM1  null deletions with the endometriosis are not consistent. Accordingly, we investigated the association between  GSTM1 ,  GSTT1  and  GSTP1  variations and susceptibility to endometriosis in Iranian women.\n\nSubjects\nIn this case-control study, 151 non-relative women with endometriosis were included, who referred to the Avicenna Infertility Clinic and Tehran Clinic Hospital, Tehran, Iran. The study protocol was approved by the Ethics Committee of the Avicenna Research Institute, and written informed consent was obtained from all participants. The diagnosis was made by visual inspection of the pelvis organs at laparoscopy, The sample size was estimated from previous study ( 15 ).\nEndometriosis women were classified to stage I to IV according to the revised American Society for Reproductive Medicine (ASRM) classification and they were found to have minimal (stage I), mild (stage II), moderate (stage III), and severe (stage IV) of endometriosis. The controls were 156 non-relative healthy women with no history of endometriosis and without any lesion as confirmed by laparoscopy. Because stage I and II of endometriosis are commonly found in asymptomatic women, therefore, in all controls absence of endometriosis was confirmed by laparoscopy ( 16 ). These people underwent laparoscopy for conditions other than endometriosis such as benign ovarian cyst.\nGenotyping\nTo investigate the association between  GSTM1 ,  GSTT1  and  GSTP1  variations and susceptibility to endometriosis in Iranian women, the genotyping was performed by multiplex PCR and PCR-RFLP. Genomic DNA was extracted by salting out procedure from 5 ml of peripheral blood samples ( 17 ). Two multiplex PCR reactions were designed for the analysis of  GSTM1  and  GSTT1  null genotypes. The  ZFX  (495 bp) and  GAPDH  (113 bp) genes were used as internal controls in multiplex reactions containing  GSTM1  and  GSTT1 , respectively. Multiplex PCR technique could not distinguish between wild homozygous and heterozygous genotype of the  GSTM1  and  GSTT1  genes.\nAccordingly, after electrophoresis the presence of  GSTM1  and  GSTT1  bands indicate that there is at least one copy of these genes. Each multiplex PCR reaction was performed in a final volume of 25 µl containing: 10 X PCR buffer, 1.5 mM MgCl2, 1U Taq DNA polymerase (CinnaGen, Iran), 0.5 mM dNTPs (Fermentas, Germany), 5 pmol of each primer, 50 ng template DNA, and sterile distilled water up to 25 µl. Amplification was performed with an initial denaturation at 94 o C for 3 min, followed by 35 cycles of amplification which was performed at 94 o C for 30 sec, 60 o C for 30 sec, 72 o C for 45 sec and a final extension at 72 o C for 5 min. The PCR products were analyzed on 1.5% agarose gels and stained with ethidium bromide. The presence of a 459 bp or 219 bp bands indicated that there is at least one copy of the  GSTT1  and  GSTM1  genes, respectively, whereas the absence of these bands indicated the null genotype for these genes ( Figure 1 ). The primer sequences and related product sizes are shown in  table I .\nThe  GSTP1  313 A/G polymorphism (rs1695) was analyzed by PCR-RFLP. The PCR amplification was carried out in a reaction mixture containing 10X PCR buffer, 2 mM MgCl2, 1U Taq DNA polymerase (CinnaGen, Iran), 0.5 mM of dNTPs, 5 pmol of each primer, 30 ng template DNA, and sterile distilled water up to 25 µl. Amplification was performed with an initial denaturation step at 94 o C for 3 min, followed by 30 cycles at 94 o C for 30 sec, annealing at 62 o C for 30 sec and extension at 72 o C for 30 sec, and a final extension at 72 o C for 5 min. The PCR products of  GSTP1  were digested with the restriction enzymes BsmAI at 37 o C overnight. The 313G allele produced two fragments with the length of 83 bp and 93 bp, while the 313A allele was not digested (176 bp). DNA fragments were subjected to 10% polyacrylamide gel electrophoresis and stained with silver nitrate ( Figure 2 ).\nStatistical analysis\nStatistical analysis was performed by IBM SPSS Version 20 (IBM Corporation, Chicago, IL, USA). Genotype and allele frequencies of each variation were compared between the case and control group by Fisher's exact test, Chi- square and logistic regression analysis. p<0.05 was considered statistically significant. All analyses were estimated by odds ratio and their 95% confidence intervals (CIs).\n\nDescriptive analysis of 151 endometriosis women and 156 controls showed that the mean age of endometriosis and control groups were 31.4±6.0 and 29.3±5.3 years old, respectively. The mean BMI in the case and control groups were 25.0±4.7 and 25.6±5.6, respectively. The  GSTM1  null genotype was significantly higher (p=0.027, OR=5.76, 95% CI:1.22-27.11) in the cases (7.3%) than the control group (1.3%).\nThis finding suggested that  GSTM1  null polymorphism may be associated with susceptibility to endometriosis. In the endometriosis group, homozygous women for the  GSTM1  null allele showed a six-fold increased risk of endometriosis than the controls ( Table II ). On the other hands, there was not a significant difference between the frequency of null and present genotype of  GSTT1  between the cases and controls ( Table II ).\nGenotype distribution in the control group for the  GSTP1  313 A/G polymorphism was in Hardy-Weinberg equilibrium (p>0.05). Genotype and allele frequencies for  GSTP1  313 A/G are summarized in  table III . Our results showed that there was significant difference in the genotype distributions of  GSTP1  313 A/G between the case and control groups. The  GSTP1  313 A/G genotype was significantly lower (p=0.048; OR=0.61, 95% CI:0.37-0.99) in the case (33.1%) than the control group (44.4%).\nPrimer sequences and their related sizes for each polymorphism\nGenotype distribution and allele frequency of the  GSTM1  and  GSTT1  polymorphisms in endometriosis women and controls\nThere is at least one copy of wild allele\nFisher’s exact test.\nGenotype distribution and allele frequency of the GSTP1 313A/G polymorphism in endometriosis women and controls\nLogistic regression (p< 0.05). n=151\nSummary of studies which evaluated the  GSTM1 ,  GSTT1  and  GSTP1  variations in endometriosis. Positive and negative results abbreviated as P and N, respectively\nResults of  GSTT1  and  GSTM1  multiplex PCR on 1.5% agarose gels. A: Lane M: Marker 100 bp; Lane 1: DDW as negative control; Lane 3:  GSTT1  null deletion (Homozygote); Lane 2, 4, 5 and 6: At least on copy of GSTT1; GAPDH: as internal control. B: Lane M: Marker 100 bp; Lane 5:  GSTM1  null deletion (Homozygote); Lane 1-4: At least one copy of  GSTM1 ;  ZFX : as internal control\nResults of RCR-RFLP for  GSTP1  313 A/G polymorphism, separated on 10% polyacrylamide gel electrophoresis. Lane M: Marker 100 bp; Lane 2 and 6: PCR product as the control for digestion; Lane 3: Wild homozygote (AA); Lane 5: Mutant homozygote (GG); Lane 1, 4 and 7: Heterozygote (AG\n\nThe aim of this study was to evaluate whether the  GSTM1 ,  GSTT1  null genotypes and  GSTP1  313 A/G polymorphism are associated with susceptibility to endometriosis. In Caucasians, the frequencies of homozygous deletions of  GSTM1  and  GSTT1  are approximately 50% and 10-20%, respectively. Most studies investigating the effect of  GSTM1  and  GSTT1  null polymorphisms do not distinguish between individuals with one or two copies of the genes; therefore, the effects of functional gene dosage could not be explored ( 17 ). The  GSTP1  313 A/G polymorphism (Ile105Val at codon 105), resulting in an enzyme with altered substrate affinity. Approximately 10% of Caucasians are homozygous for this mutation and 40% are heterozygous ( 18 ).\nOur results showed that the  GSTM1  null genotype might be associated with the risk of endometriosis in Iranian women. The endometriosis women with  GSTM1  homozygous null genotype had a six- fold increased risk of developing endometriosis (p=0.027; OR=5.76). Also,  GSTP1  313 A/G polymorphism was associated with the endometriosis, however, 313 A/G genotype had a protective effect (p=0.048, OR=0.61), which decreases the risk of the disease. In contrast, no significantly differences between the  GSTT1  null deletion and endometriosis was observed. To compare our findings, we search the PubMed database for studies that examined the association between  GSTM1 ,  GSTT1 , and  GSTP1  313 A/G polymorphisms with endometriosis up to July 2014 ( Table IV ).\nAltogether, we found 15 publications, in which nine and eight studies were performed in Caucasian and Asian populations, respectively. Briefly, positive and negative results were found in seven and six of these studies, which evaluated the  GSTM1  null genotype and endometriosis, respectively ( 18 - 23 ,  25 - 30 ). Only one out of five studies, in a Turkish population, reported that there is a positive association between  GSTP1  313 A/G and endometriosis ( 14 ,  27 ,  28 ,  30 ,  31 ). Two out of seven publications found an association between  GSTT1  null genotype and the disease ( 21 ,  23 ,  26 - 30 ). Positive association of  GSTM1  null genotypes and endometriosis in our study is consistent with the results of Hosseinzadeh  et al  which performed an association study in an Iranian population ( 21 ). In contrast, in another study in Iranian population by Seifati  et al , no association was found between this null polymorphism and endometriosis ( 32 ).\nAs  table IV  clearly shows, the results of association studies in different populations are inconsistent, which could be attributed to small sample sizes, poorly matched control group and heterogeneity within populations. Because minimal and mild stages of endometriosis may be found in asymptomatic women, therefore, in control group absence of endometriosis should be confirmed by laparoscopy ( 16 ). However, Morizane  et al  used umbilical cord blood from female newborn infants as population controls for an association study of  GSTM1  and  GSTT1  variations in women with endometriosis in a Japanese population ( 29 ). If the case and control groups are not well matched for ethnicity or geographic origin then false positive association may be occurred because of the confounding effects of population stratification ( 33 ).\nTwo meta-analysis evaluated the glutathione S-transferase variations in endometriosis women ( 13 ,  34 ). Guo  et al  performed a meta-analysis involving 14 studies investigating  GSTM1  and nine  GSTT1  studies. The results of their analysis demonstrated an association of  GSTT1  polymorphism and endometriosis, with a 29% increased risk of endometriosis in individuals homozygous for  GSTT1  null genotype. They found no evidence that women with  GSTM1  null genotype have increased risk of developing endometriosis ( 13 ). However, there was evidence of publication bias in this meta- analysis, indicating that the size of the increased risk associated with the  GSTT1  deletion variant may actually be smaller or non- existent ( 9 ). In a recent meta- analysis by Chen  et al  they concluded that the  GSTP1  313 A/G may not be associated with endometriosis risk ( 34 ).\nIn conclusion, the  GSTM1  and  GSTT1  null variations may be associated with the increased risk of endometriosis in Iranian population. Additional studies on different populations are necessary to further confirm the role of glutathione S-transferase variations in the pathogenesis of endometriosis.","source_license":"CC0","license_restricted":false}